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Method for knocking off animal FGF5 gene by using CRISPR-Cas9 system

A gene and expression system technology, applied in the field of knocking out FGF5 gene in animals using CRISPR-Cas9 expression system, can solve the problems of complicated operation, complicated design and production of ZFNs, high cost, and achieve improved transfection efficiency, wide applicability, and operation. simple effect

Active Publication Date: 2015-04-22
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0007] Although ZFNs and TALENs have the advantages of high targeting efficiency compared with traditional homologous recombination, they still have many defects, mainly including: 1. The DNA cutting domain of ZFNs and TALENs is FolkI, which must form a dimer Therefore, at least two DNA expression structures must be used for gene targeting in mammals, which will have higher requirements for transfection efficiency during cell transfection; 2. The design and production of ZFNs are relatively complicated and costly High, and it is difficult to control the cost within an acceptable range when applied to a large number of mammalian gene targeting; 3. The DNA recognition rules and design requirements of ZFNs and TALENs are relatively strict, and it may not be possible to find suitable ZFNs and TALENs recognition regions in the target gene sequence , so that it cannot be used for gene targeting; 4. When targeting different genes or the same gene of different species, it is necessary to design and construct new ZFNs and TALENs expression plasmids or mRNAs, and the operation is complicated; 5. ZFNs and When TALENs perform multi-gene targeting, it is difficult to obtain high targeting efficiency due to the limitation of the size of the carrier and mRNA molecules
There is no report on the knockout of animal FGF5 gene by CRISPR-Cas9 expression system

Method used

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  • Method for knocking off animal FGF5 gene by using CRISPR-Cas9 system

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1 Construction of CRISPR-Cas9 expression system for FGF5 gene

[0035] 1. Compare the FGF5 gene sequences of different species (human, mouse, pig, cattle, sheep, goat), find a relatively conserved region, design sgRNA in these two regions and obtain a sgRNA sequence information. The DNA sequence of the sgRNA specifically targeting the second exon of the FGF5 gene is shown in SEQ ID NO.1.

[0036] 2. Construction of pX330-F2: (1) Design and synthesize the DNA sequence of the sgRNA recognition region that recognizes the second exon of FGF5, as shown in SEQ ID NO.1; (2) The synthesized sgRNA sequence is subjected to a gradient after phosphorylation Cooling and annealing, the specific steps are to mix the synthesized oligoDNA with 10X T4Ligation Buffer and T4PNK at a ratio of 2:2:1, then add 3 times the volume of water to make up the system, then incubate at 37°C for 30min, and then denature at 95°C for 5min. Then, the temperature was lowered to 25°C at a rate of ...

Embodiment 2

[0040] Example 2 In vitro transcription

[0041] The constructed in vitro transcription vectors pIVT-F2-T and pCas9-puro3 were used for in vitro transcription mediated by T7 promoter, that is, the T7 promoter was used as the promoter of in vitro transcription, and RNA polymerase was used to realize the in vitro transcription from DNA to mRNA. The specific method of transcription process is as follows: using SalI and NotI linearized vectors pIVT-M2-T and pCas9-puro3 respectively, and then using the linearized in vitro transcription vector as a template, adding T7 transcriptase, buffer and rNTPs, and incubating at 37 °C for 6 h, Then, DNase was added at 37°C, and the template DNA was digested for 15 minutes to remove the template DNA. After the protein impurities were removed by phenol extraction, the transcribed mRNA was obtained by ethanol precipitation, and the transcribed mRNA was purified by adsorption column. The specific method was: add 3.5 Double the volume of binding bu...

Embodiment 3

[0042] Example 3 Using the CRISPR-Cas9 system for the FGF5 gene to produce gene targeting mice

[0043] 1. Pronuclear injection and embryo transfer

[0044] Take the prokaryotic fertilized eggs of B6D2F1 mice, use a microinjector to inject the pre-mixed Cas9mRNA / sgRNA mixture (the final concentration of Cas9mRNA is 150ng / μl, and the final concentration of sgRNA is 20ng / μl), and inject it into the cytoplasm of mouse fertilized eggs or in the nucleus. The injected fertilized eggs are transferred to culture medium for short-term culture, and then transplanted into the fallopian tubes of recipient mother mice to produce gene-targeted mice.

[0045] 2. Identification of gene targeting mice

[0046] After the surrogate mother mice were produced, when the offspring reached 2 weeks of age, the tails of about 1 cm were cut out, and the mouse tail genome was extracted by phenol imitation after proteinase K digestion at 55 °C. Using the mouse tail genome as a template, design primers ...

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Abstract

The invention provides a method for knocking off an animal FGF5 gene by using a CRISPR-Cas9 system. The method comprises the following steps: firstly, acquiring a DNA sequence aiming at an sgRNA recognition area of a second FGF5 exon, wherein the base sequence of the DNA sequence is as shown in SEQ ID NO.1; secondly, establishing an sgRNA expression structure of the second FGF5 exon, inserting a T7 starter before an sgRNA transcriptional start site, establishing an in-vitro transcription carrier of Cas9 protein, and regulating and controlling by using the T7 starter. Cas9 mRNA and sgRNA are obtained through the in-vitro transcription carrier of Cas9 and sgRNA, and the method can be used for knocking off the animal FGF5 gene.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering and genetic modification, and in particular relates to a method for knocking out animal FGF5 gene by using CRISPR-Cas9 expression system. Background technique [0002] Since the rise of genetic engineering in the 1980s, a large number of gene editing technologies have emerged to meet the needs of scientific research. Among them, gene targeting technology is a technology for precise and precise modification of genes in higher animals. Traditional gene targeting technology relies on spontaneous homologous recombination (HR, homologous recombination) in vivo, and the efficiency is only about 1 / 10 6 . In recent years, in order to solve the problem of low efficiency of homologous recombination, people use artificially constructed hybrid molecules to cut specific DNA sequences to improve the efficiency of gene targeting. Among them, artificial complex molecules with endonuclease as the core ar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/66C12N15/89A01K67/027
Inventor 侯健厉建伟安晓荣
Owner CHINA AGRI UNIV
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